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The result in the truncated DNA is the same. Some reagents, e.g. DMS, sometimes do not block the reverse transcriptase, but trigger a mistake at the site in the DNA copy instead. These can be detected when using high-throughput sequencing methods, and is sometimes employed for improved results of probing as mutational profiling (MaP). [14] [15]
The highest DNA adsorption efficiencies occur in the presence of buffer solution with a pH at or below the pKa of the surface silanol groups. The mechanism behind DNA adsorption onto silica is not fully understood; one possible explanation involves reduction of the silica surface's negative charge due to the high ionic strength of the buffer.
Physisorption, also called physical adsorption, is a process in which the electronic structure of the atom or molecule is barely perturbed upon adsorption. [ 1 ] [ 2 ] [ 3 ] Overview
The ratio of absorbance at 260 nm vs 280 nm is commonly used to assess DNA contamination of protein solutions, since proteins (in particular, the aromatic amino acids) absorb light at 280 nm. [ 2 ] [ 7 ] The reverse, however, is not true — it takes a relatively large amount of protein contamination to significantly affect the 260:280 ratio in ...
The DNA can then be rehydrated with aqueous low-salt solutions allowing for elution of the DNA from the beads. This method yields high-quality, largely double-stranded DNA which can be used for both PCR and RFLP analysis. This procedure can be automated [9] and has a high throughput, although lower than the phenol-chloroform method. This is a ...
A probe that hybridizes only to a single DNA segment that has not been cut by the restriction enzyme will produce a single band on a Southern blot, whereas multiple bands will likely be observed when the probe hybridizes to several highly similar sequences (e.g., those that may be the result of sequence duplication).
In the case of large DNA molecules, the DNA is frequently cut into smaller fragments using a DNA restriction endonuclease (or restriction enzyme). In other instances, such as PCR amplified samples, enzymes present in the sample that might affect the separation of the molecules are removed through various means before analysis. Once the nucleic ...
For DNA oligonucleotides, i.e. short sequences of DNA, the thermodynamics of hybridization can be accurately described as a two-state process. In this approximation one neglects the possibility of intermediate partial binding states in the formation of a double strand state from two single stranded oligonucleotides.